This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. A remarkable protein architecture has evolved to stabilize the human erythrocyte membrane and thereby enable mature cells to repeatedly withstand the shear stresses and deformations that are required as they traverse the capillaries in the peripheral circulation. Though the identity of the major proteins that comprise this architecture are known, atomic resolution structures of some isolated domains have been determined, and sparse data exist that suggest which amino acid residues are involved in the interactions, very little is currently known about the global structure of this complex of proteins. It is known that the cytoplasmic domain of the anion exchange protein (AE1;also known as band 3) serves as a critical organizing center for assembly of the major complex of proteins that stabilizes the membrane. It is also known that interactions between the cytoplasmic domain of band 3 (cdb), the membrane adaptor protein ankyrinR, and protein 4.2 are required for normal erythrocyte shape and membrane stability. Disruptions in this assembly lead to abnormally shaped erythroctyes and a condition known clinically as hemolytic anemia. Two problems that we have encountered at Vanderbilt is that DEER is not reliable for measurement of distances shorter than 20 [unreadable] and cw EPR is minimally sensitive to distances in the 18-22 [unreadable] range. Double Quantum Coherence (DQC), an alternative pulsed dipolar spectroscopy technique pioneered by the Freed group at ACERT fills in this gap very nicely and provides sensitivity to inter-probe distances to even shorter distances down to the 12 [unreadable] range. We will collaborate with the Center to make inter-probe distance measurements in the 12 to 20 [unreadable] range by DQC.